Paramyxoviruses are the leading causative agents of acute viral respiratory tract infections. Among the paramyxoviruses, human metapneumovirus (hMPV), human respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (hPIV3) account for more than 70% of acute viral respiratory diseases. Despite the enormous economic losses and emotional burdens these viruses cause, vaccines and anti- viral drugs are currently not available. The 5' end of the messenger RNA (mRNA) of paramyxoviruses contains a unique methylated cap structure that is essential for viral gene expression and, subsequently, viral replication. Available evidence suggests that formation and methylation of the paramyxovirus mRNA cap structure differs from that of their hosts. This difference could potentially be used as a target for vaccine development and anti-viral therapies. This project will be focused on hMPV, a newly discovered human pathogen, first identified in 2001. The objective of this project is to understand the mechanism of mRNA capping and methylation in hMPV and to explore methyltransferase as a potential target for the development of novel vaccines against hMPV. Our specific aims are: (1) to define the mechanism of mRNA capping in hMPV; (2) to define the mechanism of mRNA cap methyltransferase in hMPV; (3) to generate methyltransferase -defective hMPV and to determine whether methyltransferase -defective viruses are attenuated and genetically stable in cell culture; and (4) to determine whether methyltransferase -defective hMPV is attenuated and genetically stable in mice and if it can be used as a live vaccine candidate. The proposed studies will not only significantly advance our understanding of viral mRNA capping and methylation, an essential step in paramyxovirus gene expression, but will also fill in a major gap in our understanding of the role of methyltransferase in hMPV pathogenesis, specifically, our understanding of whether methyltransferase-defective hMPV can be used as a live vaccine candidates. In the long-term, our research will facilitate the rational design of new vaccines and anti-viral drugs by targeting viral mRNA cap formation.